AAB BIOFLUX

Advances in Agriculture & Botanics-

International Journal of the Bioflux Society

Antimicrobial activity of supercritical fluid

extracts of two Philippine medicinal plants,
Psidium guajava and Euphorbia hirta:
Implications to community health
1
Maria L. D. G. Lacuna, 2Marvelisa L. Carmona, 3Beverly B. Amparado,
4
Mary A. F. Daclan, 5Lourdes A. Ranido
1
Department of Biological Sciences, Mindanao State University-Iligan Institute of
Technology, Iligan, Philippines; 2 Aurora State College of Technology - Aurora Marine
Reseach and Development Institute, Aurora Province, Philippines; 3Department of
Biology, Mindanao State University, Marawi, Philippines; 4Department of Sociology -
Anthropology, Mindanao State University, Marawi, Philippines; 5Mindanao Sanitarium and
Hospital, Tibanga, Iligan, Philippines. Corresponding author: M. L. D. G. Lacuna,
mileskung@yahoo.com

Abstract The study is aimed to determine the antimicrobial activity of supercritical crude extracts of
Psidium guajava and Euphorbia hirta against four bacteria such as Staphylococcus aureus, Bacillus
subtilis, Escherichia coli, Pseudomonas aeruginosa and the two fungi namely, Aspergillus niger and
Candida albicans. Fresh leaves of P. guajava and whole plant of E. Hirta were extracted using
Supercritical Fluid Extraction at 120 atm with supercritical CO2 as the extracting solvent. Antibacterial
activities of the crude extracts against the six microorganisms were determined by Filter Paper Disc
method. Results showed that the crude extracts of the two plants exerted vary degrees of inhibition to
the six test microorganisms. P. guajava exerted pronounced inhibitory action against the gram-positive
bacteria (B. subtilis and S. aureus) but minimal against the gram-negative species (E. coli and P.
aeruginosa) and fungi (A. niger and C. albicans). E. Hirta extract showed weak bacterial activity against
all test microorganisms. Comparison in the antimicrobial activities between these two plants revealed P.
guajava to be much stronger than E. Hirta but only against gram-positive bacteria while both plants have
the same minimal inhibitory effect against gram-negative and fungi species. Further, P. guajava extract
showed stronger bactericidal effect against gram-positive bacteria when compared with the standard
antibiotic chloramphenicol which may scientifically validate its effectiveness as alternative herbal
remedies to treat ailments common in rural areas. The weak antimicrobial and antifungal actions of E.
hirta demonstrated in our study may further point to the plant’s other medicinal value such as platelet-
increasing effect as reported by some authors.
Key Words: Philippine medicinal plants, Psidium guajava, Euphorbia hirta, supercritical fluid extraction,
antimicrobial activity.

Introduction. Man has relied on most plants for its therapeutic values (aside from its
uses as food, spices and condiments in foods, timber and clothing) since the beginning of
time. In many countries in Asia, Latin America and Africa, knowledge on herbal plants for
its medicinal uses is largely dependent on one’s knowledge that have been handed over
from generation to generation. Nowadays, the role of herbal plants as alternative
medicines in treating ailments in many developing countries are gaining popularity
because of the high cost that it entails in using modern life-saving medicines,
hospitalization and primary healthcare services. Further, in developing countries where
modern healthcare facilities are inaccessible to majority of the people especially in rural
areas, the use of local herbal remedies is the main solution in treating their illnesses. In
fact, the World Health Organization (WHO) reported that more than 80% of the world’s
population now depends largely on traditional medicine for their healthcare needs. At
present, compilation of studies pointing to the importance of medicinal plants in providing

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natural source of bioactive compounds that are used mainly for medicine purposes are
increasing. In addition, literatures and ethnobotanical reports are quite available
suggesting that plants are the sleeping giants of pharmaceutical industry (Hamburger &
Hostettmann 1991; Akinpelu & Onakoya 2006).
In the Philippines, there are more than 1000 species of plants with medicinal
values, however, the Philippines’ Department of Health had approved and recommended
for use ten medicinal plants only. Out of these ten, only two plants namely, Cassia Alata
and Psidium guajava are antibacterials. Despite this information, the importance of
medicinal plants in the Philippines cannot be overlooked because most of these traditional
plants are being used as poultices (for wounds, scabies and skin diseases), liniments and
topical ointments (Penecilla & Magno 2011).
P. guajava (Family Myrtaceae) commonly known as guava or also locally known as
“bayabas” is one of the ten herbal plants recommended for use by the Philippine
Department of Health (DOH). It is widely distributed in the Philippines and is common in
backyards because it grows well in various soil types. The branches are 4-angled with
opposite leaves (Hernandez 1980; Ticzon 1997). It is a small tree that can grow up to 3
meters tall with greenish-brownish smooth bark. The round globular fruit, which starts as
a flower, is small and measures 3 to 6 cm long, and when ripe becomes soft and turns
yellowish-green (www.philippineherbalmedicine. org/bayabas.htm). As a folklore
medication in the country, P. guajava fruit, bark and leaves are used. Decoction of fresh
leaves is used as an antiseptic wash and aromatic bath, for diarrhea, mouthwash, ulcers,
wounds, scabies, and vaginal wash for post-partum care in women. Infusion of fresh
leaves is used for wound cleaning to prevent infection, for toothache, gum swelling and
nosebleeds. The bark is boiled and drank thrice a day for diarrhea and vertigo. The twigs,
chewed at the ends until frayed, are used as alternative for toothbrushing with whitening
effect (Seidenschwarz 1994; Ticzon 1997; Balongcod & Balongcod 2011; Olowa et al
2012). In the scientific field, many pharmacological investigations have demonstrated the
ability of P. guajava leaves, barks and fruits to exhibit antimicrobial, anti-diarrheal,
antimalarial, antitussive, hepatoprotective, anti-oxidant, anti-genotoxic and
antimutagenic, anti-allergic, anticancer/antitumor, cardiovascular, hypotensive, anti-
hyperglycemic, anti-inflammatory/analgesic, antinociceptive, wound healing, antipyretic,
spasmolytic and CNS depressant activities (Gutierrez et al 2008; Kamath et al 2008;
Joseph & Priya 2011; Kumar 2012). These exhibited activities were attributed to different
bioactive compounds isolated from extracts in different parts of guava. In particular, the
active flavonoid compound quercetine derivatives, terpenoids, tannins and other
glycosides found in leaf extract of P. guajava showed strong antimicrobial activities
(Arima & Danno 2002; Limsong et al 2004; Akinpelu & Onakoya 2006; El-Mahmood
2009b; Adeyemi et al 2009; Metwally et al 2010; Rattanachaikunsopon & Phumkhachorn
2010; Joseph & Priya 2011; Penecilla & Magno 2011; Banu & Sujatha 2012).
Another important medicinal plant in the Philippines is Euphorbia hirta (Family
Euphorbiaceae) commonly known as asthma weed or cat’s hair and is also locally known
as “tawa-tawa”. The plant is slender-stemmed and bushy that grows with several tiny
branches from the base to the top. It has a height of 2 inches with several tiny flowers
bunched together with opposite rhombus leaves blotched with purple and toothed at its
edge. The plant thrives in open wastelands, grasslands, pathways and roadsides
(Quisumbing 1978). As a folklore remedy in the country, the whole plant is boiled with
water and drank thrice daily to lower high fever and dengue fever (Olowa et al 2012),
whereas fresh leaves are used to cure dengue fever (Quisumbing 1978) as well as
bronchitis, asthma, constipation, dysentery, vomiting, conjunctivitis, colic, skin diseases,
thrush, snake bites, and hemostatic (www.stuartxchange.com/AltMed.html). Extract from
leaves are also applied on sore eyes while the smoke coming from the burnt leaves are
inhaled for asthma relief (Balongcod & Balongcod 2011). Several scientific evaluations on
the pharmacological properties of extract of leaves, stem, bark and whole plant have
pointed out the efficacy of E. hirta as an antimicrobial agent (Sudhakar et al 2006;
Ogbulie et al 2007; El-Mahmood 2009a; Okoli et al 2009; Kumar et al 2010b; Sharma et
al 2010; Bakkiyaraj & Pandiyaraj 2011; Singh et al 2011; Perumal et al 2012) because
of the presence of bioactive compounds such as tannins, saponins, phenolics, flavanoids,

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cardiac glycosides, anthroquinones and alkaloids. Other pharmacological activities of the
plant include antihelminthic (Adedapo et al 2005), antithrombocytopenic (Apostol et al
2012), anti-inflammatory/antiasthmatic (Ogunlesi et al 2009; Das et al 2010),
antimalarial (Ogunlesi et al 2009), antioxidant (Jagadeesan et al 2011), antidiabetic
(Kumar et al 2010a), antifungal, antidiarrheal, antifertility and antiamoebic (Tona et al
2000).
Supercritical fluid extraction is a technique that uses carbon dioxide for extraction
of natural products instead of the traditional organic solvents which are suspected to be
carcinogens. Therefore, it has the potential to be environmentally friendly “green”
processing technique because it works in closed loop and hence is not a generator of
carbon dioxide (which is a growing concern in global warming). Further, it is a potential
solvent for replacement of many undesirable organic solvents, which present a threat to
environment, health and safety in the work place (Basniwal et al 2005; Nagarsekar et al
2010).
Due to the importance and wide distribution of these two plants and the
environmental benefits in employing supercritical fluid CO2 as extracting solvent, this
study was conducted. It aims to determine the presence of antimicrobial activities of
these herbs against 4 bacteria and 2 fungi, compare the antimicrobial activities between
these two plants and then compare the effectiveness of P. guajava and E. hirta extracts
to standard antibiotics such as chloramphenicol and mycostatin.
By providing a scientific data on the sensitivity of the test organisms to the
extracts of P. guajava and E. hirta, this research can justify and promote the use and
application of these herbal preparations in rural communities where such practices are
prevalent. In particular, the extracts may prove to be all-natural, safe, effective and
inexpensive alternative to costly commercially sold drugs for infectious diseases such as
skin diseases, diarrhea and candidiasis that are commonly associated with the six test
microorganisms.

Material and Method. Four net bags filled with leaves of P. guajava and three net bags
filled with whole plants of E. hirta were collected from the botanical garden at MSU-
Marawi City. The samples were washed thoroughly and then air-dried for 12 days. The
dried samples were cut into smaller pieces and homogenized using an osterizer. About
150 grams of the homogenized sample was placed in a cloth bag and loaded in the
Supercritical Fluid Extractor machine (Figure 1) using supercritical CO2 as extracting
solvent. Experimental extraction condition was set at 120 atm at 40°C. After an hour of
extraction, the supercritical CO2 extracts (in a form of paste, Figure 2) were subjected for
antibacterial and antifungal activities by adopting the Filter Paper Disc method. The crude
extract was transferred into a small vial and was reconstituted in 2.5 ml of hexane.
Sterile filter paper disc (Advantec) with a diameter of 6 mm was impregnated with 30 µL
of the extract, and aseptically dried under a sterile hood. Subcultures of bacteria (24
hour) and fungi (48 hour) were prepared in agar slants. One loopful of the test organism
was seeded in 50 mL of melted sterile top agar. Observing aseptic measures, 15 mL of
the nutrient agar (bacterial culture medium) or potato-dextrose agar (fungal culture
medium) was dispensed into sterile petri dishes and allowed to cool for 15 minutes.
Subsequently, 2.5 mL of the seeded top agar was spread over the solidified nutrient agar
or potato dextrose agar and allowed to cool for 15 minutes. The dry discs impregnated
with the plant extracts were placed on the surface agar plates and incubated at 37oC.
Standard discs of chloramphenicol (30 µg/disc) and mycostatin (30 µg/disc) were used as
positive controls. Zones of inhibition were measured in millimeters using a ruler after 24
hours for bacterial cultures and 36 hours for fungal cultures. The antimicrobial activity of
the P. guajava, E. hirta and the two standard antibiotics against Staphylococcus aureus,
Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Aspergillus niger and Candida
albicans were recorded based on the diameter of the zone of inhibition (ZOI) exerted by
the plants against the test microorganisms. This was done by measuring the clear area
(halos) around the disc using the millimeter scale of a transparent ruler. Each experiment
was carried out in triplicates.

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 Figure 1. Supercritical Fluid Extraction (SFE) machine.

Figure 2. Plant crude extracts of (A) E. hirta and (B) P. guajava under 120 atmospheric
pressure using Supercritical Fluid Extraction (SFE).

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Results and Discussion.

Antimicrobial activity, as exhibited by the zone of inhibition, were observed in the

supercritical fluid extracts of P. guajava and E. hirta tested against the four bacteria and
two fungi (Table 1). It showed that the tested plants exerted different degrees of
antimicrobial activity against the test microorganisms.

Table 1
Antimicrobial activities of supercritical fluid extracts of P. guajava, E. hirta and standard
antibiotics

Zones of Inhibition (ZOI)

Microorganisms (mm)*
P. guajava E. hirta Chloramphenicol Mycostatin
B. subtilis 33.42 9.58 26.58 nd
S. aureus 24.83 9.67 24.75 nd
E. coli 10.58 9.17 21.92 nd
P. aeruginosa 9.75 9.00 22.75 nd
A. niger 7.92 7.75 nd 23.83
C. albicans 8.83 9.25 nd 21.92
mm* - mean of three replicates, nd - not done.

Although P. guajava extracts exerted varying degrees of sensitivity to all microorganims,

strong antibacterial activities were more pronounced against gram- positive bacteria, B.
subtilis (Figure 3 A) and S. aureus (Figure 3 B), with mean zone of inhibition of 33.42
mm and 24.83 mm, respectively, when compared to the gram-negative species (Figure 4
A, B) and fungi (Figure 5 A, B). Furthermore, antibacterial activity exerted by guava
extract towards these gram-positive bacteria were stronger than those of the standard
antibiotic chloramphenicol (ZOI: B. subtilis = 26.58 mm and S. aureus = 24.75 mm).
Our present findings are in accordance with the studies of Sanches et al (2005),
Suresh et al (2008) and El-Mahmood (2009b) who reported the leaves of P. guajava to
have considerable activity against gram-positive bacteria but less for gram-negative
species. It is suggested that the strong bactericidal activity exhibited by the leaf extracts
against the gram-positive bacteria is possibly due to substances that has protein-
degrading activity (Anas et al 2008; Akinpelu & Onakoya 2006) and can complex with
bacterial cell walls (Anju et al 2011; Akinpelu & Onakoya 2006). Bactericidal substances
usually affect the synthesis of bacterial cell wall and cell membrane resulting to leakage
of cytoplasmic content and hence, cell death (Tortora et al 2001). It has been
demonstrated that tannins and flavonoid derivatives quercitine, which are numerous in
guava leaves, have protein-binding activities (Owen & Johns 1999; Belemtougri et al
2006) and has the ability to complex with cell walls of bacteria (Cowan 1999; Akinpelu &
Onakoya 2006), respectively. Brotz-Oesterhelt et al (2005) further showed an
antibacterial compound, acyldepsipeptides (ADEPS), which inhibits gram-positive bacteria
via uncontrolled proteolysis. Ali et al (1996) also reported flavonoids to be inhibitory to
S. aureus and has been used in treatment of inflammed tissues. On the other hand,
antibacterial activity exerted by the plant extract against gram-negative bacteria was
weak when compared to those of standard chloramphenicol (Table 1). Their less
susceptibility may be attributed to their thick murine layer in the cell wall which may
have prevented the entry of most of the environmental substances such as antibiotics
and plant-based biocides into the cell (Khandhasamy et al 2008; Tortora et al 2001). In
addition to this barrier, its periplasmic space may also have enzymes which can
breakdown foreign molecules that are introduced from the outside. With this, guava
extract can be categorized as bactericidal to the two gram-positive bacteria suggesting
that the plant may contain active chemical components that can kill bacteria. In contrast,
guava extract can be categorized as bacterisotatic to the two gram-negative species
having only the ability to stop the growth or multiplication of the microorganisms but not
necessarily harming or killing them.

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Figure 3. Antimicrobial assay plate for the antibacterial activity of P. guajava on the
growth of (A) B. subtilis and (B) S. aureus showing wide zone of inhibition.

Figure 4. Antimicrobial assay plate for the antibacterial activity of P. guajava on the
growth of (A) E. coli and (B) P. aeruginosa showing minimum zone of inhibition.

The antifungal activity exhibited by the guava extract was not particularly strong when
compared to those of the standard mycostatin (Table 1) and this in agreement with the
study of Egharevba et al (2010) and Singh et al (2011) who both demonstrated guava
leaf extract in having no activity against fungi isolates. However, our results are in
contrary to the studies of Metwally et al (2010) and Anju et al (2011) who showed leaf
guava extracts having strong antifungal activity. Our present results further support the
knowledge of P. guava leaf extracts possessing strong antibacterial properties rather than
pronounced antifungal activity although their antimycotic action could not be ruled out.

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Figure 5. Antimicrobial assay plate for the antibacterial activity of P. guajava on the
growth of (A) C. albicans and (B) A. niger showing minimum zone of inhibition.

E. hirta extracts exerted minimal antimicrobial activity against the four bacteria (Figure 6
A-D) and two fungi (Figure 7 A, B) when compared to those of the standard antibiotic
mycostatin. This is evident by their minimum zone of inhibition that ranges between 9-
9.67 mm and 7.75-9.25 mm, respectively (Table 1). With this, E. hirta may be
categorized as bacteriostatic and mycostatic to the four bacteria and two fungi,
respectively.
The present study is in accordance with the study of Bakkiyaraj & Pandiyaraj
(2011) who demonstrated low antimicrobial activity exerted by the plant extract against
the fungus C. albicans. Although our findings showed bacterial effect but in mininimal
activity, it is in contrary with the studies of Mohamed et al (1996), Ogbulie et al (2007),
Suresh et al (2008), El-Mahmood (2009a) and Bakkiyaraj & Pandiyaraj (2011) who
demonstrated E. hirta leaf extracts exerting pronounced antimicrobial action against
gram-positive and gram-negative bacteria as well as the fungus A. niger (Mohamed et al
1996).
Since the present study shows that the plant extract could not provide promising
results on its antibacterial and antifungal activities, it can be suggested that the plant
extract may be promising in other activities such as its platelet-increasing effect or
antithrombocytopenic as reported by Apostol et al (2012). In the Philippines, the fresh
leaves (Apostol et al 2012) and whole plant (Olowa et al 2012) of E. hirta are traditionally
used as a cure for dengue fever.
Comparison between the inhibitory actions against these six microorganisms by
the extracts of P. guavaja and E. Hirta revealed that guava extracts are more potent
against gram-positive bacteria (Table 1). However, the minimal inhibitory actions of both
plant extracts were the same against gram-negative bacteria and fungi species.

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Figure 6. Antimicrobial assay plate for the antibacterial activity of E. hirta on the
growth of (A) B. subtilis, (B) S. aureus, (C) E. coli and (D) P. aeruginosa showing
minimum zone of inhibition.

Implications to Community Health. In the Philippine social stratification system, it is a

fact that a majority of the Filipinos belong to the lower class, many of whom could not
likely appropriate a fraction of their budget for health maintenance. The statistics
released by the National Statistics Coordination Board indicates that approximately 24
out of 100 Filipino families did not earn enough to satisfy their basic food and non-food
requirements. It is in this junction that the Department of Health strengthens its
community health programs. The use of herbal medicine is part of its health promotion in
the community level and functions in the encouragement of the Filipino populace to make
use of whatever is available within their immediate environment and in their respective
communities. As the use of locally available medicinal plants and herbs has been
advocated (many of which just grow in the household backyard and fields), their
effectiveness in the treatment of common ailments as attested to by the National Science

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Development Board (Cuevas 2007) has led to continuing research on the medicinal
properties of those already identified and promoted herbs and plants.

Figure 7. Antimicrobial assay plate for the antibacterial activity of P. guajava on the
growth of (A) C. albicans and (B) A. niger showing minimum zone of inhibition.

P. guajava is fortunately included as one of the ten approved herbal medicines of the
Department of Health (DOH) in the Philippines. E. hirta on the other hand, though not
one of those approved herbal medicines yet, has been already widely used around the
country, amongst rural and urban community dwellers alike who would be more than
willing to share their good and positive claims of the healing experiences they have had
with it; whether used independently in the home-setting or synergistically with prescribed
pharmacological medicines in the hospital setting in the form of fluid replacement
therapy. At the heart of this study is the “community,” the core of the Philippine society,
with the premise of providing a better alternative of low cost medicines but with equal
potency with those synthetic medicines available in the market nowadays. This study is
deemed to eventually motivate researchers and students to advance the restoration of
health by promoting alternatives integrated in every health teaching plan. Since
traditional use of plants with therapeutic values are encouraged as alternative medicines,
hence the proper conservation and sustainable use of such plant resources and the
incorporation of the traditional knowledge with scientific findings by local communities
should be enhanced through education.

Conclusions. The present findings on the effectiveness of P. guajava extract as strong

antimicrobial agent against gram-positive bacteria strongly supports the
traditional/folkloric knowledge of local users about their selection of this plant sample as
treatment of various ailments caused by this type of bacteria. Since our study revealed
the extract of P. guajava to be stronger than the commercially sold chloramphenicol, it
will further support the use of these plant-based medicines as an effective and strong
alternative medicine. In a country where hospitals and modern-life saving drugs are
inaccessible to the majority of the people living in rural areas, local herbal remedies that
are enhanced by scientific study can help them immensely. With this knowledge at hand,
the way to aid them is to teach them the appropriate preparation and application of these
plant-based medicines in a proper and aseptic way so as to ensure absence of secondary
infection. Moreover, the weak antimicrobial and antifungal actions of E. hirta
demonstrated in our study further point to the plant’s value in platelet-increasing effect

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or antithrombocytopenic as reported by some authors. Considering that the Philippines is
frequently experiencing bouts of dengue fever, there is therefore a need for detailed
scientific study of the plant’s medicinal uses in order to ensure and vouch for the
effectiveness and safe use of E. hirta.

Acknowledgements. The authors would like to acknowledge Dr. Roberto Malaluan of

the School of Engineering Technology, Mindanao State University-Iligan Institute of
Technology, Iligan City, Philippines for the use of the supercritical fluid extractor machine
and his research laboratory where the experiments were conducted.

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Received: 01 December 2012. Accepted: 03 January 2013. Published online: 14 January 2013.
Authors:
Maria Lourdes Dorothy Garcia Lacuna, Department of Biological Sciences, College of Science and Mathematics,
Mindanao State University-Iligan Institute of Technology, Andres Bonifacio Avenue, Iligan City 9200,
Philippines, mileskung@yahoo.com
Marvelisa Laput Carmona, Aurora State College of Technology-Aurora Marine Reseach and Development
Institute, Zabali Campus, Baler, Aurora Province, Philippines, talabaler@yahoo.com
Beverly Bermejo Amparado, Department of Biology, College of Natural Science and Mathematics, Mindanao
State University-Main Campus, Marawi City , Philippines, amparadobevs@yahoo.com
Mary Ann Faller Daclan, Department of Sociology-Anthropology, College of Social Sciences and Humanities,
Mindanao State University-Main Campus, Marawi City , Philippines, mfdaclan@gmail.com
Lourdes Alivio Ranido, Mindanao Sanitarium and Hospital, Tibanga, Iligan City 9200, Philippines,
odettetaz@yahoo.com
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution and reproduction in any medium, provided the original author and source
are credited.
How to cite this article:
Lacuna M. L. D. G., Carmona M. L., Amparado B. B., Daclan M. A. F., Ranido L. A., 2013 Antimicrobial activity of
supercritical fluid extracts of two Philippine medicinal plants, Psidium guajava and Euphorbia hirta: Implications
to community health. AAB Bioflux 5(1):1-12.

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